Quantitation of Carisoprodol and Meprobamate in Urine and Plasma Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Carisoprodol and meprobamate are centrally acting muscle relaxant/anxiolytic drugs that can exist in a parent-metabolite relationship (carisoprodol → meprobamate) or as a separate pharmaceutical preparation (meprobamate aka Equanil, others). The monitoring of the use of these drugs has both clinical and forensic applications in pain management applications and in overdose situations. LC-MS/MS is used to analyze urine or plasma/serum extracts with deuterated analogs of each analyte as internal standards to ensure accurate quantitation and control for any potential matrix effects. Positive ion electrospray is used to introduce the analytes into the mass spectrometer. Selected reaction monitoring of two product ions for each analyte allows for the calculation of ion ratios which ensures correct identification of each analyte, while a matrix-matched calibration curve is used for quantitation.

Factors affecting carisoprodol metabolism in pain patients using urinary excretion data.

Carisoprodol is a skeletal muscle relaxant prescribed to treat pain. Carisoprodol is metabolized to meprobamate, an active metabolite with anxiolytic effects, by the genetically polymorphic CYP2C19 enzyme. Concomitant use of CYP2C19 substrates or inhibitors may alter carisoprodol metabolism, with therapeutic and/or toxic implications for effectively treating patients with pain. This was a retrospective analysis of urinary excretion data collected from patients with pain from March 2008 to May 2011. Carisoprodol and meprobamate urine concentrations were measured by liquid chromatography-tandem mass spectrometry, and the metabolic ratio (MR) of meprobamate to carisoprodol concentrations was determined in 14,965 subjects. The MR geometric mean and 95% confidence interval (95% CI) of the young group (105, 95% CI = 99.1-113) were ∼47.4% higher than the middle-aged group (71.9, 95% CI = 70-73.8) and nearly two times higher than the elderly group (54.4, 95% CI = 51.3-57.6). Females had a 20.7% higher MR compared with males. No significant change in the MR was observed with overall CYP2C19 inhibitor or substrate use. However, evaluation of individual inhibitors showed co-administration with esomeprazole or fluoxetine was associated with a 31.8 and 24.6% reduction in MR, respectively, compared with controls (P < 0.05). Omeprazole did not significantly affect the MR. Patient-specific factors such as age, sex and co-medications may be important considerations for effective carisoprodol therapy.

Evaluating the relationship between carisoprodol concentrations and meprobamate formation and inter-subject and intra-subject variability in urinary excretion data of pain patients.

Using urinary carisoprodol data from pain patients, our objectives were to determine the relationship between carisoprodol concentration and its conversion to meprobamate, and quantify the intra-subject and inter-subject variability in carisoprodol metabolism. Liquid chromatography-tandem mass spectrometry was used to quantitate carisoprodol and meprobamate concentrations in urine specimens. The log creatinine-corrected carisoprodol versus log creatinine-corrected meprobamate showed a marginal positive relationship (R(2) = 0.395), with a 29.1-fold variance between subjects at the mean carisoprodol concentration. The geometric mean carisoprodol and meprobamate urine concentrations were 0.519 ± 3.38 mg and 28.2 ± 2.34 mg analyte per gram creatinine, respectively. The log metabolic ratio (MR) versus log creatinine-corrected carisoprodol displayed a marginal positive correlation. A subpopulation of outliers with higher carisoprodol and lower meprobamate levels were considered poor metabolizers and represented 0.483% (n = 21) of the study population. Using a curve-fit mathematical model, we estimated 0.318% (n = 10) to be ultra-rapid metabolizers. The inter-subject population geometric standard deviation (SD) of the MR was 3.64. The intra-subject geometric median and mean SD of the MR were 1.60 (interquartile range: 1.28, 2.07) and 1.72 ± 1.60, respectively. Inter-subject variability was 2.27 times greater than the median intra-subject variability. With a better understanding of urine carisoprodol and meprobamate concentrations and variability, urine drug testing provides a useful monitoring reference for clinicians.

Validation of a new homogeneous immunoassay for the detection of carisoprodol in urine.

The objective of this project was to validate a new high-throughput homogeneous enzyme immunoassay (HEIA) for the rapid detection of carisoprodol in human urine. Carisoprodol (Soma(®)) and meprobamate are widely prescribed as musculoskeletal pain relief drugs and are listed as one of the 10 most frequently identified drugs associated with DUI cases. Carisoprodol has a short elimination half-life of 1-3 h; however, its major active metabolite, meprobamate, has a longer elimination half-life of 6-17 h. As a result, it is important for an immunoassay to cross-react with both compounds. The advantage of this new assay is that cutoff concentrations can be adjusted between 100 and 500 ng/mL. The reportable range was 25 to 1000 ng/mL for carisoprodol and 50 to 10,000 ng/mL for meprobamate. The intraday coefficient of variation (% CV) for the semi-quantitative assay was less than 1%. The homogeneous assay was validated with a total of 86 urine samples previously analyzed by liquid chromatography-tandem mass spectrometry with carisoprodol concentrations ranging from 50 to 10,000 ng/mL. The accuracy was found to be 100% when immunoassay cutoff concentrations of carisoprodol and meprobamate were set at 100 and 1000 ng/mL, respectively.

A one-step and sensitive GC-MS assay for meprobamate determination in emergency situations.

A rapid, sensitive, and ready-to-use gas chromatography-mass spectrometry method for meprobamate assay using carisoprodol as internal standard is described. The method involves extracting a 0.2-mL sample with chloroform, previously acidified with HCl 0.2N. For the quantitative analysis, we used selected-ion monitoring mode, selecting the ion m/z 144 for quantification of meprobamate and m/z 245 for carisoprodol. Excellent linearity was found between 0 and 200 mg/L plasma. The limit of detection was 0.58 mg/L, and the limit of quantification was 1.93 mg/L. A high reproducibility [intra-assay coefficient of variation (CV) range of 2.3-4.3% and interassay CV range of 5.5-12.3%] and accuracy (intra-assay range of 96.8-112.3% and interassay range of 85.5-99.3%) were observed. Recoveries were concentration-independent (87.0%, 76.2%, and 81.2% for 20, 75 and 150 mg/L, respectively). No interference from endogenous compounds, other metabolites of meprobamate, or frequently coadministered drugs was detected. This sensitive, simple assay for meprobamate in plasma, whole blood, and urine meets the current requirements for bioanalytical assays in overdose cases.

Simultaneous determination of carisoprodol and acetaminophen in an attempted suicide by liquid chromatography-mass spectrometry with positive electrospray ionization.

An adult female ingested a considerable quantity of carisoprodol/acetaminophen tablets, which are not commercially available in Japan, in an attempt to commit suicide. Generally, because of lack of the appreciable ultraviolet absorbance or fluorescence, carisoprodol and its major metabolite meprobamate are determined by gas chromatography or gas chromatography-mass spectrometry. Complicated derivatization is, however, necessary to that methodology. Thus, we investigated the derivatization-free, highly sensitive, and simultaneous determination of carisoprodol, meprobamate, and acetaminophen by means of liquid chromatography-mass spectrometry (LC-MS) with positive electrospray ionization. A semi-micro ODS column was used. Ammonium acetate solution (10mM) and acetonitrile were used as mobile phase at a flow rate of 150 microL/min using gradient elution. MS parameters were as follows: capillary voltage, 3.5 kV; cone voltage, +30 V; extractor voltage, 5 kV; and ion source temperature, 100 degrees C. Urine samples pretreated by Oasis HLB cartridge, or plasma samples deproteinized by adding ice-cold acetonitrile were analyzed by LC-MS. The limits of quantitation for each compound were as follows: 0.50 ng/mL for carisoprodol; 10 ng/mL for acetaminophen; and 1.0 ng/mL for meprobamate. In the present case, carisoprodol and acetaminophen were the only drugs detected. Meprobamate was also found as the metabolite of carisoprodol in both urine and plasma. The plasma levels of carisoprodol, acetaminophen, and meprobamate on arrival were 29.5, 245, and 46.7 microg/mL, respectively. These levels were extremely high compared with therapeutic plasma concentrations. Despite the high plasma concentrations of these drugs, which correspond to fatal levels, the patient survived.

Carisoprodol: an unrecognized drug of abuse.

During a 6-month monitoring period, carisoprodol was detected in the urine specimens of 19 patients for whom drug screening had been ordered for purposes of patient care. The clinical history suggested that in 7 cases the drug was abused or implicated in a suicide attempt or gesture. In another 7 cases, the drug was used primarily for medical purposes, and in 5 cases the reason for use could not be determined. One patient ingested homemade tablets that were found to contain carisoprodol. In an additional case, the drug was detected in breast milk. Physical findings, clinical history, and treatment are described, and the profile of a typical carisoprodol user is discussed. It seems that carisoprodol has become an unrecognized drug of abuse, at least in our community. This drug and its metabolite, meprobamate, should be included in comprehensive drug screening.

Meprobamate overdosage: a continuing problem. Sensitive GC-MS quantitation after solid phase extraction in 19 fatal cases.

We describe a simple method for the urinary identification and blood quantitation of meprobamate suitable for any toxicological laboratory. After urinary screening using GC-MS technology, quantitation is performed by GC-MS in the selected-ion monitoring mode. Isolation of the drug is achieved by solid phase extraction on a C-18 cartridge. A specific elution is obtained by three volumes of acetone:triethylamine (99:1 v/v). Lidocaine is used as internal standard. RSDs (%) of the within-day and between-day precision studies are always less than 7.2 on the entire range of calibration. Linearity is inspected using an analysis of variance ANOVA. Homogeneity of the variances is tested using Hartley's test. Weighted linear regression is then computed. Limits of detection and quantification are given by an analysis of the blanks. The present method was applied in our laboratory over a period of 1 year. Meprobamate appeared as a drug which still has a significant frequency (5.5%) and is the most frequently involved in fatal pharmaceutical overdoses (15.3%). Post mortem concentrations ranged from 41 to 397 mg/l (mean = 182) and are compared to those of the literature.

Determination of meprobamate in human plasma, urine, and hair by gas chromatography and electron impact mass spectrometry.

A sensitive and specific quantitative assay for the determination of meprobamate in human fluids and hair is described. Meprobamate and an internal standard, vinylbarbital, are isolated by acid extraction and methylated by derivatization. The final extract is separated on a 12-m capillary column HP-1 and drugs are detected by selected ion monitoring at m/z 162 and m/z 182 for meprobamate and the internal standard, respectively. Applications in forensic science, particularly for hair analysis, are presented.

Carisoprodol concentrations from different anatomical sites: three overdose cases.

Three cases involving overdoses of carisoprodol are presented. Concentrations of carisoprodol and its major metabolite meprobamate, were determined in urine, vitreous humor, heart, and femoral blood. All drugs were quantified by gas chromatography/mass spectrometry (GC/MS).

Use of hemodialysis in meprobamate overdosage.

A case of meprobamate overdosage successfully treated with hemodialysis is described. The patient was admitted 4 hours after an overdosage of meprobamate (30-40 g) deeply unconscious, hypotensive, in respiratory failure and with a serum meprobamate level of 50 mg/100 ml. Hemodialysis was instituted using a Gambro parallel flow dialyzer and a portable re-circulating dialyzate delivery system (Redy, CCi Life Systems). Meprobamate removal with hemodialysis was 672+/-167 mg/hr with a corresponding clearance of 61.97+/-9.9 ml/min. Drug removal with forced diuresis was 177+/-23.4 mg/hr. Metabolic degradation of the drug was approximately 482 mg/hr with a plasma disappearance rate of 5.2%/hr. No drug could be detected in the dialyzate fluid after its passage through the Redy re-circulating dialyzate system. Because of the rapidity of metabolic degradation of meprobamate, we feel that hemodialysis should be reserved for severe clinical intoxication and either compromised normal excretory routes or progressive clinical deterioration.